1. Field of the Invention
The present invention relates to a KEX2 endoprotease produced by a recombinant DNA technique, a process for production thereof, a gene coding for the KEX2 endoprotease and used for the process, and the use of the KEX2 endoprotease.
2. Description of the Related Art
KEX2 endoprotease of Saccharomyces yeast is a protease which specifically processes a mating type factor and a killer factor. The properties of the KEX2 endoprotease are reported to be as following: (1) KEX2 cleaves a C-terminal of Lys-Arg sequence in a mating type factor, and a C-terminal of Lys-Arg sequence and Pro-Arg sequence in a killer factor; (2) a purification thereof was attempted, and it was found that the enzyme is present in a membrane fraction and requires calcium ion for activation thereof; (3) KEX2 is a glycoprotein having a molecular weight of 100 to 120 K Dalton; (4) KEX2 specifically cleaves a C-terminal of sequences Arg-Arg, Lys-Arg, and Pro-Arg (BBRC, 144, 807-814, 1987).
On the other hand, it is considered that, in animal hormones (biologically active peptides), the mature form thereof is generated from a corresponding precursor through the cleavage by a specific protease, and the structures of there cleavage sites very closely match a substrate specificity of the KEX2 endoprotease. For example, a mature form of insulin is generated by cleaving a precursor consisting of B chain-C peptide-A chain, to remove the C-peptide. Sequences Arg-Arg or Lys-Arg locates at the ends of the C-peptide (Nature, 282, 525-527, 1979). Further, a glucagon molecule also has a sequence Lys-Arg at both ends thereof (Proc. Natl. Acad. Sci. USA, 79, 345-349, 1982); a gastrin molecule has an Arg-Arg sequence at the C-terminal thereof (Pro. Natl. Acad. Sci. USA, 79, 1049-1053, 1982); somatostatin is located in the C-terminal side of the precursor thereof, and an Arg-Lys sequence precedes a mature somatostatin (Nature, 288, 137-141, 1980).
Note, when producing a desired peptide such as a peptide hormone by a recombinant DNA technique, it is sometimes difficult to directly produce the desired peptide due to an instability of the desired peptide in a host cell, and as one way of solving this problem, the desired peptide is produced as a fused protein comprising a protective (or stabilizing) protein, and the fused protein is cleaved to liberate the desired protein or peptide. To liberate the desired protein or peptide, however, the fused protein must be cleaved at a predetermined specific site, and since this specific cleavage site is similar to the cleavage site for the KEX2 endoprotease, it is considered that the KEX2 endoprotease will be useful for cleavage of the fused protein to liberate the desired protein or peptide.
Nevertheless, since the KEX2 endoprotease is recovered from Saccharomyces yeast in a membrane fraction and must be solubilized by a surfactant for purification, problems arise in the industrial production thereof. To solve these problems, a gene engineering technology can be advantageously utilized, and if an enzyme which is soluble exhibits the same substrate specificity as that of the native KEX2 endoprotease, this is even more advantageous.